Abstract
The ability of inhibitors selective for the type 5 phosphodiesterase isozyme (PDE5) to act on the photoreceptor PDE isozyme (PDE6, the central effector enzyme for visual transduction) is poorly understood. Because PDE5 inhibitors are currently used as therapeutic agents, it is important to assess the potency and mechanism of action of this class of PDE inhibitor on PDE6. We show that E4021 (sodium 1-[6-chloro-4-(3,4-methylenedioxybenzyl)-aminoquinazolin-2-yl]piperidine-4-carboxylate sesquihydrate) inhibits activated PDE6 (KI = 1.7 nM) as potently as PDE5. This makes E4021 the most potent inhibitor of PDE6 discovered to date. The effectiveness of E4021 to inhibit nonactivated PDE6 (with bound inhibitory γ subunits) is reduced 40-fold compared with the activated enzyme. Furthermore, at intermediate E4021 concentrations and high cGMP concentrations, nonactivated PDE undergoes activation of cGMP hydrolysis rather than inhibition. We demonstrate direct competition of E4021 and the γ subunits for binding to the catalytic site. Measurements of cGMP binding to noncatalytic regulatory sites on the catalytic subunits of PDE6 rule out an allosteric effect of E4021 by direct binding to these noncatalytic sites. We conclude that E4021 is a competitive inhibitor of cGMP hydrolysis and that the γ subunit also competes with both E4021 and substrate for catalytic site binding. An understanding of the effects of PDE5-targeted drugs on retinal PDE6 requires a knowledge of the complex interactions among substrate, drug, and inhibitory γ subunit at the catalytic site of both nonactivated and activated forms of PDE6.
Footnotes
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Send reprint requests to: Dr. Rick H. Cote, Department of Biochemistry and Molecular Biology, University of New Hampshire, Durham, NH 03824-3544. E-mail: rick.cote{at}unh.edu
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This work was supported by National Institutes of Health Grants EY-05798 (R.H.C.) and EY-10843 (N.O.A.) and by the New Hampshire Agricultural Experiment Station (Scientific Contribution 1972).
- Abbreviations:
- E4021
- sodium 1-[6-chloro-4-(3,4-methylenedioxybenzyl)-aminoquinazolin-2-yl]piperidine-4-carboxylate sesquihydrate
- PDE
- phosphodiesterase
- Pγ
- inhibitory γ subunit of type 6 phosphodiesterase
- Pαβ
- catalytic heterodimer of type 6 phosphodiesterase
- PDE5
- cGMP-binding cGMP phosphodiesterase
- PDE6
- photoreceptor phosphodiesterase
- ROS
- rod outer segments
- zaprinast
- 2-o-propoxyphenyl-8-azapurin-6-one
- IBMX
- 3-isobutyl-1-methylxanthine
- KD
- dissociation constant
- KI
- drug inhibition constant
- Pγ-1–83BC
- recombinant Pγ mutant in which residues 83–87 are deleted and replaced with a cysteine, followed by labeling with 3-(bromoacetyl)-7-diethylaminocoumarin
- Received August 19, 1998.
- Accepted November 13, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
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